Tetrathionate Reduction Production HydrogenSulfide …byboth trimethylamine oxide reduction (10, 94) andfumar-ate reduction (96). Thus, one might expect tetrathionate to serve as anelectron

Vol. 51, No. 2MICROBIOLOGICAL REVIEWS, June 1987, p. 192-2050146-0749/87/020192-14$02.00/0Copyright C 1987, American Society for Microbiology

Tetrathionate Reduction and Production of Hydrogen Sulfidefrom Thiosulfate

ERICKA L. BARRETT* AND MARTA A. CLARKDepartment of Food Science and Technology, University of California, Davis, California 95616

INTRODUCTION ........................................................................ 192

REDUCTIONS OF SULFUR COMPOUNDS PERFORMED BY NON-SULFATE-REDUCING BACTERIA ........................................................................ 193

OCCURRENCE OF TETRATHIONATE AND THIOSULFATE REDUCTION .................................193

Reactions under Consideration ........................................................................ 193

Natural Occurrence of the Substrates ........................................................................ 194

Survey of Bacteria That Reduce Tetrathionate and Thiosulfate ....................................................194

H2S from Thiosulfate versus H2S from Cysteine.................................................................. .....195Source of the confusion ........................................................................ 195

Different volatile sulfides ........................................................................ 195

Different sources of H2S................................................................6........19TETRATHIONATE AND THIOSULFATE REDUCTION BY THE SULFATE-REDUCINGBACTERIA ........................................................................ 196

TETRATHIONATE AND THIOSULFATE REDUCTION BY ENTEROBACTERIACEAE....................197

Citrobacter, Proteus, and Salmonella..................................................................... 197

Background ........................................................................ 197

Reductases ........................................................................ 197

Electron transport system ........................................................................ 198

Regulation ........................................................................ 198

Genetics ........................................................................ 199

Assimilatory thiosulfate reduction ........................................................................ 199

Other Enterobacteriaceae ........................................................................ 199

TETRATHIONATE AND THIOSULFATE REDUCTION BY THIOSULFATE-OXIDIZINGBACTERIA ........................................................................ 199

Thiobacillus-Like Isolates ......................................................................... 199

Pseudomonas aeruginosa ..................................................................... 200

CONCLUDING REMARKS ........................................................................ 200

LITERATURE CITED........................................................................ 201

INTRODUCTION

In the biological sulfur cycle as presented in most text-books of microbiology, reduced free sulfur (hydrogen sul-fide) is produced in two different ways. It is the product ofsulfate reduction by a unique group of anaerobic bacteriathat includes Desulfovibrio and Desulfotomaculum, and it isreleased from organic matter without any change in oxida-tion state during processes of protein decomposition by avariety of other microorganisms. Although many organismsassimilate sulfate and reduce it for their biosynthetic needs,dissimilatory sulfate reduction appears to be performedexclusively by the sulfate-reducing bacteria (130). Theyobtain energy by means of an anaerobic respiration in whichsulfate serves as the primary electron acceptor, althoughthey can also use partially oxidized sulfur compounds fortheir energy needs (127, 130). These bacteria are typicallyfound in mud or waterlogged soil that contains organicmatter and oxidized sulfur (138). When hydrogen sulfide isfound in other environments, it is generally assumed to bethe product of protein decomposition.

* Corresponding author.

On the other hand, it has long been known in the diagnos-tic laboratory that there are a number of bacteria that reducethiosulfate or sulfite to H2S or reduce tetrathionate tothiosulfate independently of their biosynthetic needs. Thereare, in fact, about 10 different commercial media availablefor testing H2S production, and the ability to produce H2S isa valued taxonomic character. Most of the organisms thatperform these reductions are quite familiar to us in manyrespects, yet we know very little about the biochemistry andphysiology of their sulfur reductions or their contribution tothe biological sulfur cycle.The goal of this review has been to assemble and analyze

the literature concerning tetrathionate and thiosulfate reduc-tions by bacteria outside the sulfate-reducing group in thehopes of providing a foundation for future studies of theseprocesses. The reduction of sulfur compounds by the well-characterized sulfate-reducing bacteria has been extensivelystudied and reviewed (83, 118, 119, 127-130, 138, 153, 161),and it was not our original intention to include these orga-nisms in this review except as the point of departure fordiscussions of the comparable processes in other bacteria.However, in the reviews of the sulfate-reducing bacteria,thiosulfate and tetrathionate reductions are clearly periph-

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TETRATHIONATE AND THIOSULFATE REDUCTION 193

TABLE 1. Eo' values for redox couples associated withsulfur-containing electron acceptors and other electron carriers

Redox couple (EV) Reference

02/H20 + 818 153N03/NO2 + 433 153DMSO/DMSa +160 179TMAO/TMAb + 130 58S4062-/S2032- + 170 63

+ 80 79+24 153

Fumarate/succinate + 33 153S032-/S2- -116 153S/S2- -270 153NAD/NADH -320 153S2032-/S2- + S032- -402 153

-420 58H+/H2 -414 153C02/formate -432 153S042-/SO32- -480 58

-516 153a DMSO/DMS, Dimethyl sulfoxide/dimethyl sulfide.b TMAO/TMA, Trimethylamine oxide/triemthylamine.

eral subjects. Thus, to bring the point of departure intofocus, it was necessary to include a brief discussion oftetrathionate and thiosulfate reductions performed by thesulfate-reducing bacteria.

REDUCTIONS OF SULFUR COMPOUNDS PERFORMEDBY NON-SULFATE-REDUCING BACTERIA

The sulfur compounds that have been shown to be re-duced by bacteria outside the sulfate-reducing group includetetrathionate, thiosulfate, sulfite, sulfur, and dimethylsulfoxide. The Eo' values of the redox couples associatedwith these reductions are shown in Table 1 along with thevalues for certain other couples of interest to this discussion.Many of the bacteria found to reduce one or more of theabove compounds have been tested for ability to reducesulfate, and the results have always been negative (28, 136,149, 150). This is not surprising in light of the low redox forthe sulfate/sulfite couple, which not only necessitates veryreduced growth conditions but also creates a significantenergy deficit prior to the energy-yielding reactions. Thus,sulfate reduction offers little advantage to bacteria that canuse other electron acceptors.There are conflicting reports regarding the redox potential

for tetrathionate reduction, but the reported values are allwithin a range near the fumarate/succinate couple at one endand near the trimethylamine oxide/trimethylamine couple atthe other end. Oxidative phosphorylation can be facilitatedby both trimethylamine oxide reduction (10, 94) and fumar-ate reduction (96). Thus, one might expect tetrathionate toserve as an electron acceptor for oxidative phosphorylationin the bacteria capable of reducing it to thiosulfate. Thiosul-fate reduction, in contrast, does not appear to be a likelycandidate for an anaerobic respiration that is accompaniedby oxidative phosphorylation. The low redox for thiosulfatereduction and the fact that the ability to reduce thiosulfate israrely found in the absence of the ability to reducetetrathionate as well (as will be discussed) suggest that thesignificance of thiosulfate to energy metabolism in manybacteria may be closely tied to the role of tetrathionate.

The reduction of dimethyl sulfoxide appears to be part ofthe anaerobic respiratory repertoire ofmany bacteria (5, 137,184, 185), but it is not performed by Desulfovibrio spp. (184).It has been studied in the photosynthetic bacteria (10, 94,137, 182) and in Escherichia coli (12, 13). Dimethyl sulfoxidereduction is not related to the reactions associated with H2Sproduction. Instead, it bears more resemblance to trimeth-ylamine oxide reduction (10, 94, 137). Thus, it is not withinthe scope of this review.The reduction of sulfite is the key energy-yielding reaction

of the sulfate-reducing bacteria (6, 102). However, it isperformed by very few bacteria outside of this group eventhough many bacteria can reduce sulfite in assimilatoryprocesses (118, 139). Dissimilatory sulfite reduction has beenreported for certain clostridial species (44, 49, 78), a free-living Campylobacter-like isolate (77), and, among the En-terobacteriaceae, Salmonella spp. and Edwardsiella tarda(117; M. A. Clark and E. L. Barrett, submitted for publica-tion). Sulfite and thiosulfate reductions are closely associ-ated reactions in the sulfate-reducing bacteria (138). Infor-mation regarding dissimilatory sulfite reduction by faculta-tive anaerobes is still too fragmentary for meaningful discus-sion at this time.The reduction of sulfur to sulfide seems to be confined to

very special situations. It is performed by some sulfate-reducing bacteria (11, 38), and it appears to be an importantenergy-yielding process for Desulfuromonas acetoxidans, ananaerobic acetate-oxidizing species found in the same envi-ronments as the sulfate-reducing bacteria (120). Sulfur alsoserves as an electron acceptor for the same Campylobacter-like isolate noted above to reduce sulfite (77), for "Spirillumsp. strain 5175" (177), an isolate which resembles theCampylobacter-like isolate (73), for Wolinella succinogenes(90), and for several thermophilic archaebacteria (143). Ele-mental sulfur is also reduced by Beggiatoa spp., therebyfacilitating survival during short periods of exposure toanaerobic conditions (101). None of the facultativeanaerobes that reduce thiosulfate or tetrathionate have beenreported to reduce elemental sulfur.

OCCURRENCE OF TETRATHIONATE ANDTHIOSULFATE REDUCTION

Reactions under Consideration

Tetrathionate and thiosulfate reductions are associatedwith reductases that perform the following reactions:Tetrathionate reductase-2[H] + S4062- = 2S2032- + 2H+(66, 123, 124); Thiosulfate reductase-2[H] + S2032- = H2S+ S032- (50, 51, 114). As the first reaction predicts,tetrathionate reduction results in significant acidification ofthe medium (66, 69, 123). In fact, typically, the reduction invitro is linear for only a short period because the reductaseloses activity as the pH falls (66, 69, 121). Medium acidifi-cation during tetrathionate reduction is the basis of simplequalitative tests for the occurrence of this activity or itsmutational loss in tetrathionate reducers (85). The earliestassays described for tetrathionate reduction consisted ofiodometric titrations of the thiosulfate formed in the pres-ence of physiological electron donors (123). Tetrathionatereductase has also been assayed by measuring hydrogenconsumption in an assay mixture containing hydrogenaseand benzyl viologen or methyl viologen as electron carrier(51, 87, 121) or by measuring benzyl viologen oxidation bytetrathionate (34). It is somewhat surprising that all of these

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194 BARRETT AND CLARK

assays, including titrations of thiosulfate, are possible incrude extracts of bacteria capable of thiosulfate reduction aswell as tetrathionate reduction, but the investigators reportthat thiosulfate reduction in vitro does not begin until alltetrathionate has been reduced (66, 124).

Thiosulfate reductase activity is distinct from the activityof rhodanase (thiosulfate:cyanide sulfurtransferase; EC2.8.1.1.), an enzyme present in mammalian cells and many

microorganisms, which cleaves thiosulfate nonreductivelyinto sulfur and sulfite (170). Desulfotomaculum nigrificanshas been shown to possess a rhodanase activity that isseparate from the thiosulfate reductase protein (24). Thio-sulfate reduction cannot be assayed with benzyl viologenbecause the Eo' for the thiosulfate couple is lower than thatfor benzyl viologen. However, it can be assayed by measur-ing methyl viologen oxidation (7) or by measuring hydrogenconsumption with methyl viologen as a carrier (33, 56).

TABLE 2. Tetrathionate and thiosulfate reductions and H2Sproduction by the Enterobacteriaceae and selected

representatives of other families'

Thiosulfate reductionTetra- Other reports'

Organism thionate H2S inreductionb TSIC Assay

Reaction (reference)e

Escherichia coli - - + LAS (28, 149)

Shigella spp.Salmonella typhi

S. paratyphi AS. gallinarumS. typhimurium andmost other sero-types

Citrobacter freundiiC. diversusC. amalonatica

- - - LAS (28, 149)+ + - LAS (149)

+ LAS (136)- - - LAS (28, 149)- + - TSI (117)+ + + LAS (28)

+

Klebsiella pneumoniaeEnterobacter spp.Erwinia spp.

Serratia marcescens Most +

S. liqeufaciens +All other Serratia spp. -

Hafnia alvei Some +

Edwardsiella tarda +

Proteus mirabilis and +P. vulgaris

Providencia spp. +Morganella spp. +Yersinia pestisY. enterocolitica Most -

Vibrio choleraeV. parahaemolyticus +

Aeromonas hydrophila Some XPasteurella multocida +Alteromonas +

putrefaciens

+ +

- Some +- NF

- NF- NF- NF

LAS (149)PIA (18)

- - H2S chemicalassay (150)

- NF-NF

t - + PIA and LAA(39, 48)

+ + PIA (73)

+ + LAS (28, 150)

- Some + LAS (28)- Some + LAS (28)- NF- NF

-NFF- LAA (165)

- - AH (62)+ NF

+ PIA (89)

a Genera included are those for which both tetrathionate reduction andthiosulfate reduction have been reported.

b Tetrathionate reduction reported by Richard (131), LeMinor and co-workers (86, 87), or Papavassiliou et al. (116).

c Reaction in triple sugar iron agar (TSI) as reported in Bergey's Manual(20, 73).

d Original reports of thiosulfate reduction which contradict Bergey's Man-ual or which involved tests other than triple sugar iron agar. NF, None found.

e Abbreviations: LAS, lead acetate strips; LAA, lead acetate agar (DifcoLaboratories); PIA, peptone iron agar (Difco); AH, Aeromonas hydrophilamedium (62).

Natural Occurrence of the Substrates

Thiosulfate and tetrathionate are reported to be present inmost soils except in very humid regions (142). It has beensuggested that heterotrophs are important in sulfur transfor-mations in the drier regions of the world because dry soil istoo aerobic for the sulfate-reducing bacteria (142). Thiosul-fate could easily be produced from the sulfur added asfertilizer by means of chemical reaction with oxygen (160).Thiosulfate has also been found in significant quantities incertain marine environments, particularly in aerobic/anaerobic interfaces where the oxygen content is highenough to oxidize sulfide to thiosulfate, but too low for rapidchemical thiosulfate oxidation (164). Thiosulfate, being aby-product of mammalian cysteine catabolism, is also pres-ent in urine (136).

Tetrathionate is an unstable compound (69) and thuswould not be expected to persist in soils. Its occurrence insoils may, instead, reflect the existence of natural processesof thiosulfate oxidation, either biological (140, 141, 160,162-164, 166) or chemical (142). Thiosulfate is readily oxi-dized to tetrathionate; in fact, a tetrathionate solution can beprepared by mixing a thiosulfate solution with either iodine(69) or ferric chloride (85). Thus, any environment thatcontains thiosulfate is potentially an environment suitablefor tetrathionate reduction.

Survey of Bacteria That Reduce Tetrathionate andThiosulfate

The bacteria outside of the sulfate reducers which havebeen tested for both tetrathionate and thiosulfate reductionare compiled in Table 2. The reported results for bacteriatested for either tetrathionate reduction or H2S production(but not both) appear in Table 3. The data compiled in Table2 show that the ability to produce H2S from thiosulfate isclosely associated with the ability to reduce tetrathionate.There are only three reports of H2S production from thio-sulfate in a bacterial group shown to be negative fortetrathionate reduction, and two of these reports were con-tradicted by other investigators. In contrast, the clear ma-jority of the bacteria tested were either positive for bothreductions or negative for both reductions, and about one-third were reported positive for tetrathionate reduction only.Perhaps this distribution indicates that the ability to reducethiosulfate is of little value in the absence of the ability toreduce tetrathionate. A additional feature revealed in Table2 is that there are contradictory reports concerning theability to produce H2S from thiosulfate, a problem that stemsfrom confusion regarding the source of reduced sulfur whenbacteria are tested diagnostically for the "ability to produceH2S." The reasons for the confusion are the topic of the nextsection. The practical result of the confusion is that it isimpossible to assemble an accurate list of bacteria thatreduce thiosulfate. Undoubtedly, many of the H2S produc-ers listed in Table 3 are bacteria that reduce thiosulfate to

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TABLE 3. Organismsa tested for tetrathionate reduction or H2S production

Tetrathionate reductionb H2S productionc

Positive Negative Positive Negative

Alcaligenes odorans Acinetobacter (Actinomyces) Acetobacter(A. faecalis) Corynebacterium diphtheriae Arachnia Actinobacillus(A. denitrificans) (Flavobacterium) Azotobacterd AgrobacteriumPseudomonas aeruginosa Moraxella Bacillus larvae (45) BifidobacteriumP. acidovorans Pseudomonas cepacia Bacterionema matruchotii Bradyrhizobium(P. maltophila) P. diminuta (Bacteroides) (156) Brucella melitensisVeilonella (180) P. fluorescens (Brucella abortus) B. ovisXanthobacter (73) P. picketti B. neotomae Capnocytophaga

(P. putida) B. ovis (148) CardiobacteriumP. pseudoalcaligenes Budvicia (15)' Cedeceae(P. stutzeri) Campylobactere GardnerelllaStaphylococcus (123) Clostridium, many species GluconobacterStreptococcus (123) Francisella HaemophilusXanthomonas Halobacterium Halomonas

Halococcusd KluyveraKingella indologenes Obesumbacterium(Lactobacillus plantarum) (82) ParacoccusNeisserid gonorrhoeae (35)d RahnellaPseudomonas mepthitica (104) Tatumella(Rhizobium meliloti) XanthobacterRothia dentocariosa XenorhabdusZymomonas (46)

a Genera included are those for which either tetrathionate reduction or H2S production (but not both) has been reported. Genus name only is given whenproperty is characteristic of all species tested; species reported to differ are listed separately. Names in parentheses indicate that property was not characteristicof 100%o of the isolates tested.

b Unless otherwise indicated, properties were reported by Richard (131).c Unless otherwise indicated, properties were reported in Bergey's Manual (20, 73).d Test was specific for the production of H2S from thiosulfate.Organisms were tested in triple sugar iron agar. In the case of Campylobacter, C. sputorum and C. concisus were positive in triple sugar iron agar, while

C. jejuni, C. coli, and C. fetus were positive with lead acetate but not in triple sugar iron agar.

H2S, but many may also be putrefactive bacteria incapableof thiosulfate reduction. As discussed below, one nust alsoquestion some of the results compiled in Table 2 even whenthe authors specified thiosulfate as the source of the H2S.The problem is that the source of reduced sulfur constitutinga positive test for H2S is not as easy to establish as one mightthink.

H2S from Thiosulfate versus H2S from Cysteine

Source of the confusion. There are at least four problemsunderlying confusion in the interpretation of tests for H2Sproduction. First, typical diagnostic testing uses media com-posed of varying amounts of thiosulfate and peptones.Second, peptones themselves contain varying amounts ofsulfur amino acids and partially oxidized sulfur compounds(132, 155). Third, the typical diagnostic media also fre-quently contain fermentable carbohydrates, and H2S pro-duction from thiosulfate can be "masked" during sugarfermentation (21, 22) either because it is repressed (M. A.Clark and E. L. Barrett, submitted for publication) orbecause it cannot be detected (21, 22). Surprisingly, carbo-hydrates do not interfere with the detection of H2S fromsulfite; in fact, fermentable carbohydrates are essential inmedia designed to detect sulfite, as contrasted with thiosul-fate, reduction (117, 176). Finally, different results are ob-tained depending on the indicator system used. Precipitationof a dark or brightly colored sulfide is the most commonindicator reaction. Thus, media for H2S testing may containlead, Iron, or bismuth or may be used with a strip of paperimpregnated with a lead salt or 5,5'-dithiobis-2-nitrobenzoic

acid suspended above the culture. Although the detectionmechanisms are similar in principle, in fact the results of thetests vary considerably with the choice of indicator. Asdiscussed below, the contradictory results may actuallyunderlie important differences in the origin and nature of thesulfide produced.

Different volatile sulfides. The 5,5'-dithiobis-2-nitrobenzoicacid strips were shown by McMeekin et al. (95) to detect avariety of volatile sulfides including methanethiol, dimethylsulfide, and dimethyl disulfide in addition to H2S. For thisreason, they were described as being "more sensitive" thanlead assay strips, which detect only H2S (95). However,Rodler and co-workers, who investigated H2S detection withlead acetate and iron chloride (132, 133), showed that leadacetate will react with various thiols as well as with H2S.They reported (133) that many bacteria generally regarded asH2S can give a positive "H2S test" in the presence ofcysteine when lead acetate was used as the indicator,although tnost of them were negative when iron chloride wasused in place of lead acetate. Rodler et al. attributed thepositive test with lead acetate to the volatile sulfides releasedthrough the action of decarboxylases or deaminases oncysteine and proposed that iron chloride was, in contrast,specific for H2S. Other investigators have suggested, in-stead, that iron chloride was simply less sensitive than leadacetate strips (21, 28, 39, 44, 82, 165, 186), but Rodler et al.(132) reported that iron chloride detected H2S producedfrom thiosulfate sooner than did lead acetate. Unfortunately,the literature contains many contradictions regarding theability to produce H2S even among investigators who allused lead acetate. A possible explanation for the contradic-

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tions is that, although lead acetate reacts with many sulfides,it is most sensitive to H2S, and the definition of positiveversus negative results depends on whether or not a true H2Sproducer is used as the positive control against which tojudge the weaker reactions. The concentration of cysteine inthe test medium is also a factor; bacteria generally regardedas H2S' yield a positive reaction with a far lower cysteineconcentration than required to obtain a positive test forbacteria generally regarded as H2S (28, 109). There arefewer contradictions among investigators who used ironchloride as the indicator, but there are also bacteria that areconsistently negative in such tests that have been shown toproduce H2S from cysteine in biochemical assays, as will bediscussed below.

Different sources of H2S. Many investigators have at-tempted to distinguish between the detection of H2S fromcysteine, i.e., cysteine desulfhydrase activity, and H2S fromthiosulfate, i.e., thiosulfate reductase activity (62, 97,149-151). Cysteine desulfhydrase catalyzes the formation ofH2S, ammonia, and pyruvate from cysteine, and it is foundin many organisms (30, 47, 72). It is induced by cysteine (30,97, 151) and is thought to protect against cysteine toxicity(30). In Salmonella spp., induction requires cysteine con-centrations of >0.1 mM, and the enzyme is repressed bysulfide at concentrations of >0.1 mM (30). The enzyme inProteus spp. was shown to be much more active underaerobic conditions than under anaerobic conditions (151).Thiosulfate reduction, in contrast, is favored underanaerobic or microaerophilic conditions (33, 34, 62, 89, 104).

In the testing of bacteria for the ability to reduce thiosul-fate, it is essential to differentiate H2S produced throughthiosulfate reduction from H2S produced via cysteine degra-dation. It is often assumed that H2S detected in complexmedia lacking added thiosulfate constitutes a test for cyste-ine degradation, and many bacteria tested in such media arespecified as "H2S' from cysteine" in the current Bergey'sManual (73). However the protein digests in complex mediacan contain thiosulfate (132, 155). Furthermore, one cannotassume that the enzymatic production of H2S from cysteineis necessarily independent of the pathway for thiosulfatereduction. In fact, the ability to reduce thiosulfate stronglyinfluences the results of tests for H2S from cysteine. Forexample, both Salmonella typhimurium (H2S' from thiosul-fate) and E. coli (H2S from thiosulfate) contain cysteinedesulfhydrase (45), but only S. typhimurium appears H2S' ina medium consisting of gelatin, nutrient broth, and ironchloride (74, 80). Similarly, Proteus vulgaris (H2S' fromthiosulfate) and Serratia marcescens (H2S- from thiosulfate)were shown to produce equal amounts of H2S from cysteinein assays (150), but only P. vulgaris appears H2S' in amedium containing peptone, cysteine, and iron chloride(133). It is interesting that Tilley, in 1923, recommendedthiosulfate as an ingredient for testing H2S production notbecause its reduction represented a taxonomic characteristicdistinct from the production of H2S from cysteine, butbecause it "improved" the distinction between H2S' andH2S species (154, 155).Both E. coli and Serratia spp. do give positive tests in the

peptone-plus-cysteine medium if lead acetate rather thaniron chloride is used as the indicator (133). One possibleexplanation for the failure to detect the H2S with iron salts isthat a reduction of the iron is a prerequisite for a positivereaction with iron chloride and only the species that canproduce H2S from thiosulfate are capable of reducing theiron. When iron is used, it is usually present as a ferric salt,either because it was added in that form or because the

ferrous salt was autoclaved in a neutral medium. A positivetest for H2S based on the formation of iron sulfide, i.e.,ferrous sulfide, requires both the reduction of ferric iron andsulfide precipitation. In contrast, the lead in lead acetatedoes not need to be reduced. We have found that E. coli doesappear H2S' in the gelatin medium if filter-sterilized ferrouschloride is added to the autoclaved medium (M. A. Clark andE. L. Barrett, unpublished data).

TETRATHIONATE AND THIOSULFATE REDUCTIONBY THE SULFATE-REDUCING BACTERIA

Much less is known about thiosulfate and tetrathionatereduction by the sulfate-reducing bacteria than is knownabout their reduction of sulfate and sulfite. Postgate (125)was the first to report that thiosulfate and tetrathionate couldserve as electron acceptors for sulfate-reducing bacteria. Hedemonstrated that a resting cell suspension of Desulfovibriodesulfuricans (a strain now designated as Desulfovibriovulgaris) quantitatively reduced thiosulfate or tetrathionateto sulfide with molecular hydrogen as reductant (125). Thatsulfide was produced from both compounds indicates thatthe reduction of tetrathionate and thiosulfate constitutes atributary of sulfite reduction. Later it was shown that lactatealso served as an in vivo electron donor (57). In a cell-freeextract, thiosulfate reduction with H2 proceeded very slowlyin the absence of the artificial electron carrier methylviologen (56). Similarly, it has since been shown that thepurified thiosulfate reductases from D. gigas andDesulfotomaculum nigrificans do not reduce thiosulfate tosulfide when reduced nicotinamide adenine dinucleotide(NADH), NADPH, reduced glutathione, or cysteine is pro-vided as electron donor in place of reduced methyl viologen(51, 100).

Thiosulfate reductase has been purified from D. vulgaris(4, 50), D. gigas (51), and Desulfotomaculum nigrificans(100). The enzyme from D. gigas also catalyzes the reduc-tion of tetrathionate to thiosulfate, using benzyl viologen aselectron donor (51), but tetrathionate reductase as such hasnot been purified from this organism or any other sulfate-reducing bacteria. The thiosulfate reductases from all threeorganisms were found in the soluble fraction (4, 50, 51, 100,107), and the enzyme has been shown in D. vulgaris to befunctionally localized on the cytoplasmic side of the cyto-plasmic membrane (7). The molecular weight of the thiosul-fate reductase from D. vulgaris (Miyazaki F strain) (4) was87,000, and that from D. gigas was determined to be 220,000(51). The optimum pH was generally in the slightly alkalinerange: it was 7.2 to 7.9 for the D. vulgaris (Miyazaki F)enzyme (4), 8.0 to 9.0 for the D. vulgaris (Hildenborough)enzyme (50), and 7.4 to 8.0 for the D. gigas enzyme. Theenzyme from Desulfotomaculum nigrificans was reported tofunction equally well at all pH values from 6.0 to 8.0. The Kmfor thiosulfate was determined for the D. gigas (0.5 mM) andDesulfotomaculum nigrificans (130 mM) enzymes. The sur-prisingly high latter value suggests that thiosulfate reductionis not a primary pathway for energy metabolism in thisorganism.

Electron transport from H2 to thiosulfate has been inves-tigated in several species of Desulfovibrio. Cytochrome C3(molecular weight, 13,000) can function as the sole electroncarrier between the hydrogenase and thiosulfate reductaseactivities in D. vulgaris (4, 126) and D. desulfuricans (19).Studies with purified hydrogenase and thiosulfate reductasefrom D. gigas showed that the most efficient electron trans-port system (other than methyl viologen) consisted of both

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cytochromes C3 and CC3 (molecular weight, 26,000) (51).Ferredoxin and flavodoxin both strongly stimulated thiosul-fate reduction in cell-free extracts of D. gigas (52; L.Guarraia, E. J. Laishley, N. Forget, and H. D. Peck, Jr.,Bacteriol. Proc., p. 133, 1968), but neither appeared to beinvolved in thiosulfate reduction by D. vulgaris (4, 181).

Thiosulfate reductase has been proposed to be part of an

enzyme system for a three-step reduction of sulfite in whichtrithionate and thiosulfate are intermediates (42, 43, 147),although there is now convincing evidence that thiosulfate isonly a by-product of sulfite reduction when sulfite is inexcess (60) and free intermediates are not normally producedduring the reduction of sulfite to sulfide (27). It is unlikelythat thiosulfate reduction alone supports growth of adeno-sine triphosphate formation because the growth yields forD.vulgaris on H2 with thiosulfate are equivalent to yieldsobtained with sulfite (6, 102). This finding suggests, instead,that thiosulfate is simply a source of sulfite.

Thiosulfate has also been shown to facilitate growth on

lactate by D. baculatus (135), D. thermophilus (134), D.africanus (59), Desulfotomaculum ruminis (29), andThermodesulfobacterium commune (183). The enzyme sys-

tems involved have not been examined. Among these orga-

nisms, only D. africanus was tested for growth withtetrathionate and was found negative (59). Thiosulfate alsosupports the growth of Desulfobulbus propionicus on

propionate (175) and Desulfobacter postgatei on acetate(152, 173). In contrast, Desulfotomaculum acetoxidansgrows only with sulfate as electron acceptor (174). In theirexamination of 92 isolates of sporulating and nonsporulatingsulfate-reducing bacteria, Skyring et al. (139) found thatmost strains grew on lactate plus thiosulfate. Thus, thiosul-fate reduction appears to be a common adjunct to theenergy-yielding systems of the sulfate-reducing bacteria.

TETRATHIONATE AND THIOSULFATE REDUCTIONBY ENTEROBACTERIACEAE

Citrobacter, Proteus, and Salmonella

Background. Citrobacter, Proteus, and Salmonella are themajor H2S-producing and tetrathionate-reducing genera ofthe Enterobacteriaceae. Reports of H2S production byProteus and Salmonella spp. go back into the clinical liter-ature of the 19th century (see reference 136 for discussion ofearly observations). Studies of H2S production in Proteusspp. by Tarr in 1933 (150, 151) and later by Mitsuhashi andMatsu in 1953 (97) demonstrated that there were two mech-anisms of H2S production, one associated with thiosulfatereduction and the other involving only cysteine. Tarr (150)pointed out that many bacteria were capable of the lattertype of H2S production, while thiosulfate reduction was a

more limited property. Mitsuhashi and Matsu (97) reportedthat formate was a major electron donor for thiosulfatereduction, while it did not have much effect on the produc-tion of H2S from cysteine, and that the two substratesinduced specifically the respective enzyme systems for H2Sproduction. More than 30 years elapsed before any furtherstudies of thiosulfate reduction by Enterobacteriaceae were

published except in the context of studies focused ontetrathionate reduction or on anaerobic respiratory systemsthat happen to share some features with thiosulfate reduc-tion.

Tetrathionate reduction was first described in 1942 by

Pollack et al. (124), who were investigating the selectiveaction of tetrathionate in selective media for Salmonella spp.They showed that tetrathionate reduction was a stoichio-metric conversion of tetrathionate into thiosulfate (123)which was dependent on an electron donor such as a sugar orformate (123) and could be induced by tetrathionate inwhole-cell preparations (70). They also found that tetrathion-ate improved yields of Salmonella spp. growing in complexmedia (68, 69) and suggested that tetrathionate was servingas an electron acceptor (68, 69, 124). Interest in tetrathionatereduction was apparently not contagious as more than 20years elapsed before any further reports were published. Therenewed interest was sparked by the possible usefulness oftetrathionate reduction as a taxonomic character that waseasily tested (85-87, 116) and by the work of LeMinor,Pichinoty, and co-workers (88, 121), who showed that thetetrathionate reduction shared many features with nitratereduction. About the same time, Stouthamer and co-workersbegan a study of tetrathionate and thiosulfate reduction inProteus, while Kapralek and co-workers examinedtetrathionate reduction in Citrobacter freundii. The com-bined work of the latter two groups of investigators consti-tutes the vast majority of the literature concerningtetrathionate and thiosulfate reduction by the Enterobacte-riaceae.

Reductases. Tetrathionate reductase was shown to bemembrane bound in both Proteus mirabilis (112, 113) and C.freundii (105). The enzyme has been partially purified fromP. mirabilis and shown to have a molecular weight of 133,000and two subunits of molecular weights 43,000'and 90,000(112, 113). The enzyme activity in C. freundii has beenpartially characterized (66, 106, 121). The Km for tetrathion-ate was 2 mM (121), and the pH optimum for tetrathionatereduction was 8.0(66, 105), with a 50% drop in activity whenthe pH drops below 7.2 (105). This is somewhat surprising inlight of the acidification that accompanies the reduction oftetrathionate to thiosulfate (66, 69, 123). Possibly the con-centrations of tetrathionate ordinarily encountered or han-dled by these bacteria is typically low or the environments inwhich tetrathionate is found are well buffered. The formerpossibility is supported by the observation that tetrathionatereductase in C. freundii is inhibited by concentrations oftetrathionate of >5 mM (66).A striking feature of the purified enzyme from P. mirabilis

is its catalytic versatility; it was found to catalyze, inaddition to tetrathionate reduction, thiosulfate and trithion-ate reduction as well as sulfide oxidation (113). Reductasefunction by the enzyme purified from P. mirabilis appears tobe tightly regulated, as thiosulfate reduction was reportednot to begin until all tetrathionate had been reduced (112,113). This may explain why the thiosulfate formed fromtetrathionate was not reduced by whole cells of C. freundii(63, 66) or Salmonella spp. (123, 124). Similarly, it has beenshown that the concentration of tetrathionate in tetrathion-ate broth determines whether or not Salmonella spp. willproduce H2S during growth; if the concentration exceeds 30mM, acidification kills the cells before the tetrathionate isconsumed and thiosulfate reduction never commences (69).Oltmann and Stouthamer (113) showed that the thiosulfateformed from tetrathionate stimulated tetrathionate reductionrather than thiosulfate reduction by the purified enzyme.

Thiosulfate reduction by P. vulgaris has been shown toinvolve only the sulfane sulfur (93). In S. heidelberg, bothsulfur atoms are reduced, but the sulfane sulfur is reducedentirely to H2S before reduction of the sulfonate sulfurbegins (93). Presumably, sulfonate sulfur reduction repre-

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sents the activity of an anaerobic sulfite reductase; S.typhimurium, but not P. vulgaris, produces H2S from sulfiteas well as thiosulfate (Clark and Barrett, submitted). Thepresence of the latter activity may explain why S. typhi-murium mutants defective in the biosynthetic reduction ofsulfite can grow without cysteine under anaerobic condi-tions, while they behave as cysteine auxotrophs underaerobic conditions (9).Oxygen was shown to interfere with tetrathionate reduc-

tion by extracts of C. freundii (65, 121). Kapralek andPichinoty (65) attributed the effect to the preferential transferof electrons to oxygen rather than to a direct effect of oxygenon the enzyme because cyanide prevented the inhibition,and the inhibition was reversible.The tetrathionate/thiosulfate reductases are membrane

bound (105, 112, 121). Studies of tetrathionate reduction bya number of Enterobacteriaceae have shown that this activ-ity, like nitrate and trimethylamine oxide reductions, isabsent in pleiotropic chlorate-resistant mutants defective inthe processing of molybdenum (32, 33, 75, 88, 111, 145;D. L. Riggs and E. L. Barrett, Abstr. Annu. Meet. Am. Soc.Microbiol. 1983, K246, p. 218). Molybdenum has beendemonstrated in tetrathionate reductase from P. mirabilis(110) and S. typhimurium (53).

Electron transport system. Although coupling of oxidativephosphorylation to tetrathionate reduction has not beendemonstrated directly, growth yield studies suggest that itdoes occur (63, 68, 146). Kapralek (63) calculated that 1.3mol of adenosine triphosphate could be formed per mol oftetrathionate reduced. Stouthamer and Bettenhausen (146)reported a P/2e ration of 0.2 for tetrathionate reduction in P.mirabilis. Thiosulfate, in contrast, was found not to improveanaerobic growth yields of P. mirabilis (146). However, theresults with thiosulfate are difficult to evaluate in light of thefact that the growth medium contained glucose, which hasbeen shown to repress thiosulfate reductase in S.typhimurium (Clark and Barrett, submitted).Formate, hydrogen, and NADH have all been shown to

serve as electron donors for tetrathionate reduction (71, 105,106, 114, 123). Novotny and Kapralek (106) reported thatsuccinate, lactate, malate, and glycerol were, in contrast,not efficient electron donors for in vitro tetrathionate reduc-tion by C. freundii, although Stouthamer apid Bettenhausen(146) reported that lactate and glycerol can supportanaerobic growth of P. mirabilis in the presence oftetrathionate. Formate is the only donor that has been shownto reduce thiosulfate (71). The pathway by which formatereduces thiosulfate may be different from that associatedwith tetrathionate reduction because hydrogen gas isevolved simultaneously with thiosulfate reduction, while nogas is evolved during reduction of tetrathionate by formate(71).Both Novotny and Kapralek (106) and Stouthamer and

co-workers (34, 114) reported that extracts of cells inducedfor tetrathionate reductase contain type b cytochromeswhich can be reduced with formate and partially reoxidizedby tetrathionate. de Groot and Stouthamer (34) found thatthiosulfate also reoxidized the same b cytochromes. Whenreduced, the Citrobacter cytochrome had an absorbancepeak at 560 nm at 200C (106). Two peaks were found informate-reduced extracts of P. mirabilis: 561 and 563.5 nm at150C. On the other hand, an essential role for cytochromes intetrathionate or thiosulfate reduction has not been demon-strated and is actually contraindicated by other findings. InP. mirabilis, tetrathionate oxidized only 15% of the type bcytochromes reduced by formate (33). Furthermore, on

media designed to indicate tetrathionate reduction (85),Salmonella heme mutants formed colonies indistinguishablefrom wild-type colonies (Clark and Barrett, unpublisheddata). The heme mutants, however, did lack the ability toreduce thiosulfate to H2S as well as methyl viologen-linkedthiosulfate reductase (Clark and Barrett, submitted), whichcould indicate that thiosulfate reductase is a heme-containing enzyme or that cytochromes are neccessary forthiosulfate reduction.Quinones were implicated in electron transfer to

tetrathionate in C. freundii (106), but were not identified.The Citrobacter tetrathionate reductase has also been shownto accept electrons from reduced flavin mononucleotide, butnot from NADH or NADPH (121). S. typhimurium mena-quinone mutants fail to reduce thiosulfate (75, 76), butproduce almost as much acid on the tetrathionate test media(85) as does the wild type (Clark and Barrett, unpublisheddata), which suggests that menaquinones are necessary forthiosulfate, but not tetrathionate, reduction. Ubiquinonemutants resembled the wild type with respect to both thio-sulfate and tetrathionate reduction (Clark and Barrett, un-published data); thus, ubiquinones are probably not involvedin electron transport to tetrathionate and thiosulfate by S.typhimurium. Clearly, the information available at this timeregarding electron carriers is too incomplete to postulate anelectron transport scheme for tetrathionate or thiosulfatereduction.

Regulation. Several aspects of the regulation of tetrathion-ate reduction are consistent with an overall picture ofrespiratory control in which there is a hierarchy of electrontransport pathways, each of which represses all systemswith lower energy potential and is, similarly, repressedduring operation of the higher-energy-potential systems. Inaccordance with this picture, neither tetrathionate nor thio-sulfate reduction occurs in the presence of oxygen (33, 65,112, 123, 145) or nitrate (2, 34). Oxygen has also been shownto prevent expression of P-galactosidase activity in Mudl(lac) insertion mutants affected in the production of H2Sfrom thiosulfate (3; Clark and Barrett, submitted). Further-more, tetrathionate reduction prevents hydrogen evolutionvia the formate hydrogenlyase system (33, 34, 71, 122), whilethiosulfate reduction, a reaction of very low redox potential,can occur simultaneously with hydrogen evolution (34, 71).Oxygen per se is reported to repress the synthesis oftetrathionate reductase (33, 34, 65), but it does not inhibitenzyme activity; however, electron transport to oxygenreversibly inhibits the activity in vitro (65, 121). On the otherhand, both inhibition and repression by nitrate occur onlywhen nitrate respiration is possible (2, 34, 145). In P.mirabilis it has been shown that tetrathionate reductasesubunits are still present in the membrane during repressionby nitrate respiration, but they are not active (111). Simi-larly, formate hydrogenlyase has been found in extracts oftetrathionate-grown cells, even though no hydrogen wasbeing produced.There are conflicting reports regarding the induction of

tetrathionate and thiosulfate reductases by their substrates.Both activities are reported to be constitutive in P. mirabilisgrown under anaerobic conditions in the absence of nitrate(33), while tetrathionate is reported to induce tetrathionatereductase in anaerobic cultures of Citrobacter (121) andSalmonella (123, 124) species. A variety of reduced sulfurcompounds, including thiosulfate, will induce thiosulfatereductase activity in S. typhimurium growing anaerobicallyin minimal medium (Clark and Barrett, submitted). Thiosul-fate reductase activity in anaerobic cultures of C. freundii is

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undetectable (66, 121) even though C. freundii do producehydrogen sulfide from thiosulfate (73). A major difficulty inresolving the conflicting reports is that many of the earlystudies used media containing glucose, and both reductasesare subject to catabolite repression (64; Clark and Barrett,submitted). Catabolite repression of thiosulfate reductase inS. typhimurium is quite severe; 5.6 mM glucose abolishes allenzyme activity in cells grown in minimal medium (Clarkand Barrett, submitted). Glucose repression of tetrathionatereductase in Citrobacter is somewhat less pronounced;activities in glucose-grown cells are about 15% of those insuccinate-grown cells.

Genetics. Very little is known about the genetics of thio-sulfate and tetrathionate reduction. Casse and co-workers(25) isolated a Salmonella mutant defective specifically intetrathionate reduction and mapped the mutation. The mu-tant was not examined further. Voll and co-workers (167,168) identified a gene essential for the production of H2Sfrom thiosulfate in the vicinity of the his operon. Theyshowed that this gene could be expressed in E. coli, althoughE. coli strains carrying this gene on an F' episome producedless H2S than did the Salmonella parent (167). They foundthat E. coli strains with extended deletions in the vicinity ofhis did not produce H2S when the episome from S.typhimurium was introduced, suggesting that there are genesin the same region present in both E. coli and S. typhimuriumwhich are required for H2S production (167).

Assimilatory thiosulfate reduction. That both S. typhi-murium and E. coli assimilate thiosulfate (37, 84, 99) whileonly S. typhimurium produces H2S from thiosulfate suggeststhat, in Salmonella sp., the dissimilatory and assimilatoryreductions of thiosulfate are distinct. However, there aresome differences between the two organisms regarding thio-sulfate assimilation which may provide clues regarding pos-sible common components of the assimilatory and dissimi-latory reactions in Salmonella spp. In S. typhimurium,thiosulfate assimilation involves the activity of 0-acetylserine sulfhydrylase B. This enzyme catalyzes theformation of S-sulfocysteine from 0-acetylserine and thio-sulfate (98, 99). It is encoded by cysM (54, 99). Although theactivity of this enzyme with sulfide, with which it synthe-sizes cysteine as opposed to S-sulfocysteine, is great enoughto permit wild-type growth rates on sulfate (55), the primary0-acetylserine sulfhydrylase activity which functions in cys-teine synthesis is that of O-acetylserine sulfhydrylase A (54,55), the product of cysK. The cysK and cysM genes arelocated adjacent to and on opposite sides of cysA; cysAencodes sulfate permease, which also serves as thepermease for thiosulfate (36, 108). Although CysK- CysM+mutants are prototrophs, CysK+ CysM- mutants areprototrophic only aerobically and are bradytrophs underanaerobic conditions (40, 54). This suggests that 0-acetylserine sulfhydrase B is required for rapid cysteinesynthesis under anaerobic conditions. Furthermore, al-though the various genes constituting the cysteine regulon,including cysM, require a functional cysB gene (the positiveregulator) for induction under aerobic conditions, cysB mu-tants behave as prototrophs under anaerobic conditions (9).Thus far, only O-acetylserine sulfhydrase A has been de-tected in E. coli (41, 172). Aerobic/anaerobic differences incysteine requirement have not been noted in E. coli. It willbe interesting to see whether the aerobic/anaerobic differ-ences in cysteine biosynthesis in Salmonella spp. and theexistence of an enzyme for the specific incorporation ofthiosulfate are actually reflections of the enzyme activitiesassociated with dissimilatory thiosulfate reduction. As noted

below, the same question also emerges in the study ofreductions performed by Pseudomonas aeruginosa.

Other Enterobacteriaceae

Hydrogen sulfide is also produced from thiosulfate by thenewly described Enterobacteriaceae genus Budvicia (15)and by Edwardsiella tarda (73). Edwardsiella tarda alsoreduces tetrathionate (73), but there have been no studies ofthese reductions in either group. Edwardsiella tarda may beespecially interesting because it is the only member of theEnterobacteriaceae family other than Salmonella that alsoproduces H2S from sulfite (117).Hydrogen sulfide-producing variants of E. coli have been

isolated on numerous occasions (8, 16, 31, 80, 81, 91, 92,115, 144, 169). In several cases, the ability to producehydrogen sulfide was shown to be plasmid mediated (23, 80,81, 91, 115, 144), although the nature of the plasmid differedfrom isolate to isolate. The plasmids also carried genes forraffinose utilization (23, 91, 115) in some cases and fortetracycline resistance (61, 144) in others. The plasmidguanine-plus-cytosine content was shown in one case to beabout 40% (81) and in another case to be 50% (14), whichsuggests that the E. coli strains may have received the abilityto produce hydrogen sulfide from diverse donors. One of theH2S' variants was tested for the ability to reduce tetrathion-ate and was found to be negative (61). In many of thereports, hydrogen sulfide production was tested in commer-cial triple sugar iron agar which contains 1% (wt/vol) lactose.This is interesting in light of the fact that lactose-positiveSalmonella spp. appear H2S in this medium, due to the"masking" of the H2S character by fermentation (22). Ap-parently, regulation of plasmid-mediated thiosulfate reduc-tion in E. coli differs from the regulation observed inSalmonella spp.There are also reports of variants of Shigella sonnei (1),

Klebsiella pneumoniae (17), and an otherwise H2S speciesof Citrobacter diversus (18), that reduce thiosulfate to H2S,but the isolates were not examined further. Trelcavan et al.(157) warned that some of the reports of H2S production bystrains of H2S species might be mistaken; they showed thatsome "H2S+" E. coli isolates were actually mixed culturescontaining the anaerobe Eubacterium lentum, which pro-duces H2S and which can grow under semianaerobic condi-tions if cocultured with certain facultative anaerobes.

TETRATHIONATE AND THIOSULFATE REDUCTIONBY THIOSULFATE-OXIDIZING BACTERIA

Thiobacillus-Like Isolates

The utilization of thiosulfate as an electron donor foraerobic growth is well documented among a number ofbacteria and is generally associated with the genusThiobacillus. Tetrathionate is also metabolized by many ofthe same organisms (67, 159), and some investigators haveproposed that tetrathionate is an intermediate in the oxida-tion of thiosulfate (166), although there is also strong evi-dence to the contrary (67). Anaerobic tetrathionate metabo-lism of such organisms was first reported by Trudinger (159),who observed that brief anaerobic incubation "sparked" theaerobic oxidation of tetrathionate by Thiobacillus sp. strainX. His studies of the anaerobic metabolism of tetrathionate(159) and trithionate (158) suggested that the substrates wereoxidized in the same way as under aerobic conditions andthat the oxidation of these compounds was most active

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under microaerophilic conditions. The interpretation ofthese studies is complicated by the nonenzymatic reactivityof tetrathionate under the conditions used (67).

Thiosulfate-oxidizing heterotrophs which actually reducetetrathionate to thiosulfate under anaerobic conditions havesince been isolated and examined. What is interesting aboutthese studies is that they revealed the existence of reversibleenzymes catalyzing both thiosulfate oxidation andtetrathionate reduction. Trudinger (160) described a"Thiobacillus-like" tetrathionate reducer isolated from soil.Because aerobic growth with thiosulfate resulted in theinduction of the tetrathionate reductase, he suggested thatthe same enzyme might be responsible for both thiosulfateoxidase and tetrathionate reductase. He was unable todemonstrate a physiological role for either activity as thio-sulfate did not improve aerobic yields and the organismwould not grow anaerobically with tetrathionate as an elec-tron acceptor.More recently, Tuttle and Jannasch (162, 164) clearly

demonstrated the existence of a reversible thiosulfateoxidase/tetrathionate reductase in "marine heterotroph16B," a different isolate which was found to respond to bothcompounds. Strain 16B was isolated from an aerobic thio-sulfate enrichment (164). Thiosulfate was shown to stimulateits aerobic growth (163), and both tetrathionate and thiosul-fate were found to support anaerobic growth on pyruvate orlactate (164). Nitrate also supported anaerobic growth (164).A soluble constitutive thiosulfate oxidase was purified fromaerobically grown cultures, and it was found to catalyzetetrathionate reduction as well (162, 171). The optimum pHfor thiosulfate oxidation was 6.0 to 6.5 and that fortetrathionate reduction was 7.5. That the Km for thiosulfate(0.15 mM) was lower than that for tetrathionate (about 4mM) and that thiosulfate inhibited tetrathionate reductionsuggested that the primary function of this enzyme wasprobably thiosulfate oxidation (162). Thiosulfate was foundto stimulate the aerobic growth of this organism undercertain conditions, although it did not seem to be a primaryenergy source (163).No other tetrathionate reductase activity was found in

aerobic cells, but anaerobic cultures grown with tetrathion-ate contained an additional tetrathionate reductase (162,171). This anaerobically induced tetrathionate reductase wasfound in the membrane fraction (171). Oxygen reversiblyinhibited its activity (162). Membrane preparations frominduced cells also contained a type b cytochrome withabsorption peaks at 414, 537, and 570 nm. A thiosulfatereductase activity was copurified with the inducibletetrathionate reductase (171), suggesting that, as withProteus spp., the two reductions are carried out by the sameenzyme.

Pseudomonas aeruginosa

P. aeruginosa is not usually classified among the thiosul-fate-oxidizing bacteria, but Starkey reported in 1934 that itoxidized thiosulfate to tetrathionate in a manner similar tothe "Trautwein" thiobacilli which had been described asThiobacillus-like heterotrophs (140). In the same experi-ments, Thiobacillus novellus and T. thioparus oxidized thio-sulfate to sulfate rather than to tetrathionate, indicating thatthe thiosulfate oxidations in P. aeruginosa and the otherheterotrophs did not represent the same enzyme systems aswere found in the autotrophs (141). P. aeruginosa is alsocapable of tetrathionate reduction (123). Although it hasbeen reported to be H2S from thiosulfate (149), it does

precipitate iron sulfide in several commercial media used totest H2S production in the clinical laboratory (Barrett,unpublished data).Chambers and Trudinger (26) demonstrated that P. aeru-

ginosa, like S. typhimurium, can synthesize S-sulfocysteinefrom thiosulfate. It seems unlikely that the reaction is centralto cysteine synthesis in P. aeruginosa because cysteine inthe growth medium did not repress activity, and hydrolysisof S-sulfocysteine into cysteine was not detected. Extractswith S-sulfocysteine synthase activity were found to oxidizeNADPH in the presence of reduced glutathione and S-sulfocysteine, a reaction which would be expected to yieldcysteine and sulfite as products (26). This observation is veryinteresting because it suggests a mechanism for the dissim-ilatory reduction of thiosulfate to sulfide by P. aeruginosa orS. typhimurium in which cysteine (derived from thiosulfateby means of the S-sulfocysteine synthase) would be thesource of sulfide released as H2S. The involvement ofcysteine as the substrate for the enzyme that releases H2Sduring thiosulfate reduction might help to explain whythiosulfate-reducing bacteria produce H2S from cysteinemore consistently than do bacteria which contain onlycysteine desulfhydrase (as was discussed above). The thio-sulfate reducers would contain two enzymes which catalyzethe formation of H2S from cysteine. The existence of anenzyme in thiosulfate-reducing bacteria that releases H2Sfrom cysteine but is distinct from cysteine desulfhydrasewould also help to explain the curious results of Guarnerosand Ortega (47) regarding the stoichiometry of the cysteinedesulfhydrase in extracts of S. typhimurium, namely, theproduction of more H2S than pyruvate from cysteine.

P. aeruginosa is ideal for future studies of thiosul-fate/tetrathionate reactions in the pseudomonads because itsgenetic system is well developed, and much is known aboutthe biochemistry of its energy metabolism.

CONCLUDING REMARKS

Tetrathionate and thiosulfate reduction by the non-sulfate-reducing bacteria may be fundamentally different from theanalogous reactions in the sulfate reducers. The latter bac-teria reduce tetrathionate and thiosulfate completely tosulfide in a process associated with soluble enzymes andtype c cytochromes that also participate in the reduction ofsulfite. It would appear, then, that the reductions oftetrathionate to thiosulfate and thiosulfate to sulfite in thesulfate-reducing bacteria constitute a branch leading to thecentral energy-yielding reaction, namely, the reduction ofsulfite. In contrast, among other microorganisms, the abilityto reduce tetrathionate and thiosulfate is far more commonthan the ability to reduce sulfite. Furthermore, thereductases are membrane bound, and the cytochromes in-duced during tetrathionate and thiosulfate reduction are ofthe b type.A role for tetrathionate reduction and thiosulfate reduc-

tion in the absence of sulfite reduction is somewhat difficultto imagine. The problem is that tetrathionate, althoughenergetically favorable, is quite unstable, and thus likely tobe rare, and thiosulfate, although more common, offers littlein the way of energetic advantage, especially to organismsthat can use other electron acceptors. Most of the bacteriathat reduce tetrathionate also reduce nitrate. Many investi-gators who have studied tetrathionate reduction havepointed out similarities between the two reductions such asthe common role of formate as electron donor (33, 71, 88),the common dependency on chl gene function for enzyme

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biosynthesis (32, 33, 88, 110, 145), the common anaerobicexpression (88, 114), and the possible common associationwith type b cytochromes (106, 111, 112). However, suchsimilarities are not sufficient justification for stuffingtetrathionate and thiosulfate reduction into a mold that wasshaped around nitrate reduction without considering othermechanisms and roles for tetrathionate and thiosulfate re-duction based on the unique features of these two electronacceptors.One striking unique feature is the ease with which thiosul-

fate and tetrathionate are interconverted and the catalyticversatility of the enzymes associated with their metabolism.Perhaps the metabolism of these compounds involves cyclicmechanisms which enable the cell to generate oxidizing orreducing power depending on cellular needs regarding regen-eration of electron carriers or processing of nutrients such asmetal cofactors. The extreme pH changes associated withthe metabolism of tetrathionate might also be used to advan-tage in the manipulation of the proton gradient across the cellmembrane.A feature of thiosulfate reduction that is not characteristic

of nitrate reduction is the production of an extremely toxicproduct, namely, H2S. Volatile sulfides have been shown tocontribute to the pathogenicity of certain oral bacteria byrendering the mucosa more permeable (103, 156). PerhapsH2S production by the Enterobacteriaceae contributes totheir ability to colonize tissues in other regions of the body.Our knowledge of the mechanisms of H2S production by

bacteria which do not reduce sulfate is still too scanty topostulate a role for these organisms in global sulfur transfor-mations. However, the widespread occurrence of the abilityto reduce tetrathionate and thiosulfate does suggest thatthese reductions are significant to the survival of manybacteria which do not rely on the sulfite produced for theirenergy needs.

LITERATURE CITED1. Aikawa, T., and H. Iida. 1977. H2S-positive Shigella sonnei.

Microbiol. Immunol. 21:289-293.2. Akuratny, O., and F. Kapralek. 1975. Inhibition of tetrathion-

ate synthesis and activity by nitrate respiration. Folia Micro-biol. (Prague) 20:69.

3. Aliabadi, Z., F. Warren, S. Mya, and J. W. Foster. 1986.Oxygen-regulated stimulons of Salmonella typhimurium iden-tified by Mu d(Ap lac) operon fusions. J. Bacteriol. 165:780-786.

4. Aketagawa, J., K. Kobayashi, and M. Ishimoto. 1985. Purifica-tion and properties of thiosulfate reductase from Desulfovibriovulgaris, Miyazaki F. J. Biochem. (Tokyo) 97:1025-1032.

5. Ando, H., K. Mitsuru, T. Karashimado, and H. Iida. 1957.Diagnostic use of dimethyl sulfoxide reduction test with En-terobacteriaceae. Jpn. J. Microbiol. 1:335-338.

6. Badziong, W., and R. K. Thauer. 1978. Growth yields andgrowth rates of Desulfovibrio vulgaris (Marburg) growing onhydrogen plus sulfate and hydrogen plus thiosulfate as the soleenergy sources. Arch. Microbiol. 117:209-214.

7. Badziong, W., and R. K. Thauer. 1980. Vectorial electrontransport in Desulfovibrio vulgaris (Marburg) growing on hy-drogen plus sulfate as sole energy source. Arch. Microbiol.125:167-174.

8. Barbour, E. K., N. H. Nabbut, and H. M. Al-Nakhli. 1985.Production ofH2S by Escherichia coli isolated from poultry: anunusual character useful for epidemiology of colisepticemia.Avian Dis. 29:341-346.

9. Barrett, E. L., and G. W. Chang. 1979. Cysteine auxotrophs ofSalmonella typhimurium which grow without cysteine in ahydrogen/carbon dioxide atmosphere. J. Gen. Microbiol.115:513-516.

10. Barrett, E. L., and H. S. Kwan. 1985. Bacterial reduction of

trimethylamine oxide. Annu. Rev. Microbiol. 39:131-149.11. Biebi, H., and N. Pfennig. 1977. Growth of sulfate-reducing

bacteria with sulfur as electron acceptor. Arch. Microbiol.112:115-117.

12. Bilous, P. T., and J. H. Weiner. 1985. Dimethyl sulfoxidereductase activity by anaerobically grown Escherichia coliHB101. J. Bacteriol. 162:1151-1155.

13. Bilous, P. T., and J. H. Weiner. 1985. Proton translocationcoupled to dimethyl sulfoxide reduction in anaerobically grownEscherichia coli HB101. J. Bacteriol. 163:369-375.

14. Bouanchaud, D. H., R. Hellio, G. Bieth, and G. H. Stoleru.Physical studies of a plasmid mediating tetracycline resistanceand hydrogen sulfide production in Escherichia coli. Mol. Gen.Genet. 140:355-359.

15. Bouvet, 0. M. M., P. A. D. Grimont, C. Richard, E. Aldova, 0.Hausner, and M. Gabrehelova. 1985. Budvicia aquatica gen.nov., sp. nov.: a hydrogen sulfide-producing member of theEnterobacteriaceae. Int. J. Syst. Bacteriol. 35:60-64.

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Tetrathionate Reduction Production HydrogenSulfide …byboth trimethylamine oxide reduction (10, 94) andfumar-ate reduction (96). Thus, one might expect tetrathionate to serve as anelectron